Multiple labeling of proteins

Fluorescent dyes are often used to label proteins before analysis by capillary electrophoresis. Fluorescent labeling produces spectacular improvements in sensitivity compared with UV absorbance detection of the native protein. However, labeling of the protein can lead to significant band broadening. This band broadening is interpreted as a result of multiple labeling of the protein, wherein one or more fluorescent molecules are bound to the protein. The heterogeneous reaction products, which are presumed to have different mobilities, generate a broad peak during electrophoresis. There has been little direct evidence for multiple labeling as the cause of band broadening of proteins. In this paper, we perform electrophoresis on native green fluorescence protein, along with the reaction products produced by fluorescence labeling. For short incubations, a series of regularly spaced components are resolved by free-zone electrophoresis; upon longer incubation, the product peaks merge together, forming a broad envelope.